Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre

Chen, Liane; Anton, Martina; Graham, Frank L.

doi:10.1007/bf02369439

Cited by 91 publications

(58 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…39 Upon infection of a 293-derived cell line that stably expresses the bacteriophage P1 Cre recombinase, 63 the packaging signal is excised from almost all the helper virus DNA, rendering it unpackageable. The helper virus DNA retains the ability to replicate and provides all of the functions required in trans for the replication and packaging of a hdAd.…”

Section: Introductionmentioning

confidence: 99%

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration

1999

View full text Add to dashboard Cite

We have developed a new helper adenovirus (Ad) based expression in mouse liver after intravenous injection of the on serotype 2, Ad2LC8cCARP, for use in the Cre/loxP sysAd2-based hdAd showed that the vector could efficiently tem (Parks et al. Proc Natl Acad Sci USA, 1996; 93: transduce the liver, and produce high levels of a foreign 13565-13570) to generate Ad vectors deleted of all protein transgene, similar to those expressed by the hdAd genercoding sequences (helper-dependent Ad vectors (hdAd) ).ated with the Ad5 helper virus. Mice immunized with hdAd2 A comparison of Ad2LC8cCARP and our original helper produced Ad2-neutralizing antibodies, which did not crossvirus (based on serotype 5, Ad5LC8cluc) showed that the react with hdAd5. To determine if successful repeat Ad two helper viruses amplified hdAd with a similar efficiency, vector administration could be achieved by sequential use and resulted in a similar yield and purity after large-scale of alternative Ad serotypes, we injected mice with hdAd2 preparation of vector. In vitro, the resulting hdAd2 had a (hSEAP) followed 3 months later by a lacZ-expressing similar transduction efficiency and expression kinetics of hdAd of either the same or different serotype. Repeated transgene (␤-gal) as the hdAd5. An important feature of administration of hdAd2 resulted in a 30-to 100-fold the helper-dependent system is that all virion components, reduction in transgene expression compared with naive except the virion DNA, derive from the helper virus. Conseanimals. In contrast, no decrease in transgene expression quently, vectors produced with help from Ad2LC8cCARP was observed when the second vector was of a different were not neutralized by antibodies against Ad5, and vecserotype. These results demonstrate that effective vector tors produced with Ad5 helper were resistant to neutralizreadministration can be achieved by the sequential use of ing antibodies against Ad2. Analysis of transgene hdAds based on alternative serotypes.

show abstract

Section: Introductionmentioning

confidence: 99%

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration

1999

View full text Add to dashboard Cite

show abstract

“…The human breast cancer cell lines MCF-7, MDA-MB-468 and MDA-MB-231 were maintained in either Dulbecco's modified Eagle's medium or RPMI. The Ad5 E1-transformed human embryonic kidney cell line 293 57 and its derivative 293Cre4 58 were maintained in minimal essential medium. All growth media (Invitrogen, Burlington, ON, Canada) were supplemented with 10% fetal bovine serum (Sigma-Aldrich Co., St Louis, MO, USA), 2 mM L-glutamine, 100 U ml À 1 penicillin and 100 mg ml À 1 streptomycin.…”

Section: Materials and Methods Cellsmentioning

confidence: 99%

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice

et al. 2012

View full text Add to dashboard Cite

Despite the tremendous potential of adenovirus (Ad) as a delivery vector for cancer gene therapy, its use in clinical settings has been limited, mainly as a result of the limited infectivity in many tumors and the wide tissue tropism associated with Ad. To modify the tropism of the virus, we have inserted the epidermal growth factor-like domain of the human heregulin-a (HRG) into the HI loop of Ad5 fiber. This insertion had no adverse effect on fiber trimerization nor did it affect incorporation of the modified fiber into infectious viral particles. Virions bearing modified fiber displayed growth characteristics and viral yields indistinguishable from those of wild-type (wt) virus. Most importantly, HRG-tagged virions showed enhanced infection of cells expressing the cognate receptors HER3/ErbB3 and HER4/ErbB4. This was significantly reduced in the presence of soluble HRG. Furthermore, HER3-expressing Chinese hamster ovary (CHO) cells were transduced by the HRG-modified virus, but not by wt virus. In contrast, CHO cells expressing the coxsackie-Ad receptor were transduced with both viruses. However, infection of an in vivo breast cancer xenograft model after intratumoral injection was similar with both viruses, suggesting that the tumor microenvironment and/or the route of delivery have important roles in infection of target cells with fiber-modified Ads.Cancer Gene Therapy (2012) 2,3 In addition to being among the most extensively studied viral vectors, Ads can be easily and economically manipulated, maintained and propagated to produce high titer stocks. 4,5 Moreover, they have the capacity to carry relatively large fragments of exogenous DNA into a wide range of cell and tissue types. 4,5 Subgroup C Ads (exemplified by the most widely used Ad5 and Ad2) attach to and enter host cells in a two-step process. 6 The initial recognition/attachment step is mediated by interactions between the knob component of fiber protein on the Ad capsid and the extracellular domain of the coxsackieadenovirus receptor (CAR) on the host cell surface. 7-11 This attachment is followed by a second interaction between the RGD motif in the penton base of the virus capsid and a v b 3 or a v b 5 cell surface integrins (secondary Ad receptors), which allows viral entry via receptor-mediated endocytosis. [12][13][14][15] Both the primary and secondary Ad receptors are expressed on a wide range of tissues leading to the observed broad tissue tropism of Ads. 16,17 Unfortunately, a large number of tumor cells appear relatively refractory to Ad infection because of limited surface expression of the primary Ad receptor. 1,[18][19][20][21][22][23] This observation has been a driving force behind recent intensive efforts to generate Ad viral vectors with altered tropism.

show abstract

“…The cell line 293Cre4 (ref. 29) is a courtesy of Dr Stefan Kochanek, University of Ulm, Germany. All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U ml -1 penicillin, 100 mg ml -1 streptomycin and 2 mM L-glutamine.…”

Section: Cell Lines and First-generation Adenovirusesmentioning

confidence: 99%

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

et al. 2011

View full text Add to dashboard Cite

Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (C) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which C is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers 410 10 infectious units and o0.1% HV contamination. The residual HVs lacked C and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of

show abstract

Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre

Cited by 91 publications

References 37 publications

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

Contact Info

Product

Resources

About